A: Infrared Absorption 197K: undried.

B: To 5 mL of water in a test tube add 2 drops of ammonium thiocyanate TS and 2 drops of ferric chloride TS, and mix. To the deep red solution add about 50 mg of Edetate Disodium, and mix: the red color is discharged, leaving a yellowish solution.

C: It responds to the flame test for Sodium 191.

Mobile phase— Add 10 mL of 1.0 M tetrabutylammonium hydroxide in methanol to 200 mL of water, and adjust with 1 M phosphoric acid to a pH of 7.5 ± 0.1. Transfer the solution so obtained to a 1000-mL volumetric flask, add 90 mL of methanol, dilute with water to volume, mix, pass through a filter having a 0.5-µm or finer porosity, and degas.

Cupric nitrate solution— Prepare a solution containing about 10 mg of cupric nitrate (Cu(NO3)2) per mL.

Standard stock solution— Transfer about 100 mg of nitrilotriacetic acid, accurately weighed, to a 10-mL volumetric flask, add 0.5 mL of ammonium hydroxide, and mix. Dilute with water to volume, and mix.

Resolution solution— Transfer 10 mg of Edetate Disodium to a 100-mL volumetric flask, add 100 µL of Standard stock solution, dilute with Cupric nitrate solution to volume, and mix. Sonicate, if necessary, to dissolve.

Standard solution— Transfer 1.0 g of Edetate Disodium to a 100-mL volumetric flask, add 100 µL of Standard stock solution, dilute with Cupric nitrate solution to volume, and mix. Sonicate, if necessary, to dissolve.

Test solution— Transfer 1.0 g of Edetate Disodium to a 100-mL volumetric flask, dilute with Cupric nitrate solution to volume, and mix. Sonicate, if necessary, to dissolve.

Chromatographic system (see Chromatography 621)— The chromatograph is equipped with a 254-nm detector and a 4.6-mm × 15-cm column that contains packing L7. The flow rate is about 2 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.35 for nitrilotriacetic acid, 0.65 for copper, and 1.0 for edetate; and the resolution, R, between nitrilotriacetic acid and copper is not less than 3. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The response of the nitrilotriacetic acid peak obtained from the Test solution does not exceed the difference between the nitrilotriacetic acid peak responses obtained from the Standard solution and the Test solution: not more than 0.1% of nitrilotriacetic acid is found.

Assay preparation— Dissolve about 5 g of Edetate Disodium, accurately weighed, in about 100 mL of water contained in a 250-mL volumetric flask, add water to volume, and mix.

Procedure— Place about 200 mg of chelometric standard calcium carbonate, previously dried at 110 for 2 hours, cooled in a desiccator, and accurately weighed, in a 400-mL beaker, add 10 mL of water, and swirl to form a slurry. Cover the beaker with a watch glass, and without removing the latter, add 2 mL of 3 N hydrochloric acid from a pipet. Swirl the contents of the beaker, and dissolve the calcium carbonate. Wash down the sides of the beaker, the outer surface of the pipet, and the watch glass with water, and dilute with water to about 100 mL. While stirring the solution, preferably with a magnetic stirrer, add about 30 mL of the Assay preparation from a 50-mL buret. Add 15 mL of 1 N sodium hydroxide and 0.30 g of hydroxy naphthol blue, and continue the titration with the Assay preparation to a blue endpoint. Calculate the weight, in mg, of C10H14N2Na2O8 in the portion of Edetate Disodium taken by the formula: in which 336.21 and 100.09 are the molecular weights of edetate disodium and calcium carbonate, respectively; W is the weight, in mg, of calcium carbonate; VT is the volume, in mL, of the Assay preparation; and V is the volume, in mL, of the Assay preparation consumed in the titration.